Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0068—General culture methods using substrates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0603—Embryonic cells ; Embryoid bodies
  • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/30—Synthetic polymers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/50—Proteins

Definitions

• the present disclosure relates to functionalized cell binding peptides and their use in preparing cell culture articles. More particularly, the disclosure relates to synthetic surfaces and articles for supporting the culture of undifferentiated stem cells in chemically defined medium.
• Therapeutic cells cells which may be introduced into a human for the treatment of disease, are being developed.
• therapeutic cells include pluripotent stem cells such as human embryonic stem cells (hESCs) which have the ability to differentiate into any of the three germ layers, giving rise to any adult cell type in human body.
• hESCs human embryonic stem cells
• This property of stem cells provides a potential for developing new treatments for a number of serious cell degenerative diseases, such as diabetes, spinal chord injury, heart diseases and the like.
• hESC-based treatments Obtaining and maintaining adequate numbers of therapeutic cells in cell and tissue culture and ensuring that these cells do not change in unwanted ways during cell culture are important in developing and controlling therapeutic cell cultures.
• stem cell cultures such as hESC cell cultures are typically seeded with a small number of cells from a cell bank or stock and then amplified in the undifferentiated state until differentiation is desired for a given therapeutic application.
• the hESC or their differentiated cells are currently cultured in the presence of surfaces or media containing animal- derived components, such as feeder layers, serum, or Matrigel TM available from BD Biosciences, Franklin Lakes NJ.
• animal-derived components such as feeder layers, serum, or Matrigel TM available from BD Biosciences, Franklin Lakes NJ.
• a functionalized peptide comprising a cell adhesive peptide which contains a cell binding sequence, at least one polymerization moiety wherein the polymerization moiety is an ⁇ , ⁇ un-saturated group or ethylenically unsaturated group which is, for example, acrylate, methacrylate, acrylamide, methyacrylamide, maleimide, fumarates or epoxides, and a spacer moiety wherein the spacer moiety is a polyethylene oxide, Xaa n where Xaa is independently any amino acid and where n is an integer from 0 to 3, from 0 to 6, from 0 to 10, from 0 to 20 or from 0 to 30, or combinations, and wherein the polymerization moiety is bound to the cell adhesive peptide or the spacer moiety.
• the cell adhesive peptide comprises the sequence:
• GGGQKCIVQTTSWSQCSKS (SEQ ID NO:2); KYGLALERKDHSG (SEQ ID NO:3); YGLALERKDHSG (SEQ ID NO:4); KGGSINNNRWHSIYITRFGNMGS (SEQ ID NO:5); GGSINNNRWHSIYLTRFGNMGS (SEQ ID NO:6); KGGTWYKIAFQRNRK (SEQ ID NO: 7); GGTWYKI AFQRNRK (SEQ ID NO: 8); KGGTSIKIRGTYSER (SEQ ID NO:9); GGTSIKIRGTYSER (SEQ ID NO: 10); KYGTDIRVTLNRLNTF (SEQ ID NO: 11); YGTDIRVTLNRLNTF (SEQ ID NO:12); KYGSETTVKYIFRLHE (SEQ ID NO:13); YGSETTVKYIFRLHE(SEQ ID NO: 14); KYGKAFDITYVRLKF (SEQ ID NO: 15);
• a cell culture article comprising a functionalized peptide covalently linked to a hydrophilic polymeric base material, wherein the functionalized peptide is described by the formula: R m - S p - C ap .
• R is a polymerization moiety, an ⁇ , ⁇ unsaturated group or ethylenically unsaturated group which includes acrylate, methacrylate, acrylamide, methyacrylamide, maleimide, fumarate, or epoxide, which is capable of polymerizing in the presence of an external energy source such as UV or visible light with an optional catalyst, or by thermal polymerization with an optional catalyst, "m" is an integer greater than or equal to 1.
• S p is a spacer.
• Sp may be a polyethylene oxide having the formula (O-CH 2 CHR')m2 where R' is H or CH3 and m2 is an integer from 1 to 20.
• Sp may be polyethylene glycol (PEG) or polypropylene glycol (PPG).
• the polyethylene oxide spacer may be of any length.
• S p may be PEG 2 , PEG4, PEG 6 , PEGs, PEG10, PEGi 2 or PPG 2 , PPG 4 , PPG 6 , PPG 8 , PPGi 0 , PPGi 2 Or PPG 20 .
• Sp may be an amino acid Xaa n where Xaa is any amino acid and n is an integer from 0 to 30, from 0 to 20, from 0 to 10, 0 to 6, or from 0 to 3, or combinations of Xaa and polyethylene oxide.
• Xaa n may comprise a lysine, glysine, glutamic acid, serine, aspartic acid or arginine amino acid, which may be a terminal amino acid.
• Xaa n is a hydrophilic amino acid.
• Xaa is lysine and n is greater than 1.
• the spacer S p may comprise polyethylene oxide spacer and amino acid spacer in any combination.
• the polymerization moiety may attach to the spacer, S p through the polyethylene oxide, through the side chain of an amino acid such as lysine or at the N-terminus of the amino acid.
• Amino acid Xaa n may be acetylated and/or amidated to protect it from degradation. However, if Xaa n is acetylated, the polymerization moiety cannot be bound to Xaa n through the N- terminus of the amino acid.
• C ap is a peptide or polypeptide which has a cell binding or cell adhesive sequence.
• the hydrophilic polymeric base material comprises hydrophilic monomers.
• the hydrophilic monomers are, for example, N- Tris(hydroxymethyl)acrylamide (ACRYLNTRIS-OH) and copolymer, glyceryl monomethacrylate (GLY-METH), poly(serine)methacrylate and copolymer (SER- METH).
• copolymers are formed by cross- linking with the following di- functional moieties which are acrylamides and/or acrylates: N,N'-(1,2- dihydoxyethylene)bisacrylamide and glycerol 1,3-diglycerolate diacrylate.
• the cell culture article has a contact angle of less than 50°.
• the spacer S p is PEO 4 .
• a method for making a cell culture surface comprising the steps of: providing a hydrophilic base material comprising hydrophilic monomers to a substrate surface; polymerizing the hydrophilic base material; providing a functionalized peptide to the surface of polymerized or cured hydrophilic base material; polymerizing the functionalized peptide to the hydrophilic base material; and, optionally washing.
• the hydrophilic base material comprises N-Tris(hydroxymethyl)acrylamide (ACRYLNTRIS-OH), glyceryl monomethacrylate (GLY-METH), poly(serine)methacrylate (SER-METH), hydroxyethylmethylacrylate (HEMA), or acrylamide (ACRYL) polymers and copolymers and optionally hydrophilic crosslinking materials such as, N,N'-(1,2- dihydroxyethylene)bisacrylamide, glycerol 1,3-diglycerolate diacrylate, or combinations thereof.
• ACRYLNTRIS-OH N-Tris(hydroxymethyl)acrylamide
• GLY-METH glyceryl monomethacrylate
• SE-METH poly(serine)methacrylate
• HEMA hydroxyethylmethylacrylate
• ACRYL acrylamide
• the functionalized peptide is described by the formula:
• Rm is a polymerization moiety, an ⁇ , ⁇ unsaturated group or ethylenically unsaturated group which includes acrylate, methacrylate, acrylamide, methyacrylamide, maleimide, fumarate, or an epoxide.
• Rm is capable of polymerizing for example, in the presence of an external energy source such as UV or visible light with an optional catalyst, or by thermal polymerization with an optional catalyst.
• "m" is an integer greater than or equal to 1.
• S p is a spacer.
• S p may be a polyethylene oxide including for example polyethylene glycol (PEG) or polypropylene glycol (PPG).
• S p may be PEG 4 .
• S p may be an amino acid Xaa n where Xaa is any amino acid and n is an integer from 0 to 30, from 0 to 20, from 0 to 10 or from 0 to 3, or combinations of Xaa and polyethylene oxide.
• Xaa n may comprise a lysine or arginine amino acid, which may be a terminal lysine or arginine.
• the spacer S p may comprise polyethylene oxide spacer and amino acid spacer in any combination. The polymerization moiety may attach to the spacer, S p through the polyethylene oxide, through the side chain of an amino acid such as lysine or at the N-terminus of the amino acid.
• the polymerization moiety may attach to the cell adhesive peptide C ap through the side chain of an amino acid such as lysine or at the N-terminus of the amino acid.
• Amino acid Xaa n or C ap may be acetylated and/or amidated to protect it from degradation. However, if Xaa n is acetylated, the polymerization moiety cannot be bound to Xaa n through the N-terminus of the amino acid.
• C ap is a peptide or polypeptide which has a cell binding or cell adhesive sequence.
• the cell adhesive peptide is:
• GGGQKCIVQTTSWSQCSKS (SEQ ID NO:2); KYGLALERKDHSG (SEQ ID NO:3); YGLALERKDHSG (SEQ ID NO:4); KGGSINNNRWHSIYITRFGNMGS (SEQ ID NO:5); GGSINNNRWHSIYITRFGNMGS (SEQ ID NO:6); KGGTWYKIAFQRNRK (SEQ ID NO: 7); GGTWYKI AFQRNRK (SEQ ID NO: 8); KGGTSIKIRGTYSER (SEQ ID NO:9); GGTSIKIRGTYSER (SEQ ID NO: 10); KYGTDIRVTLNRLNTF (SEQ ID NO: 11); YGTDIRVTLNRLNTF (SEQ ID NO: 12); KYGSETT VKYIFRLHE (SEQ ID NO: 13); YGSETT VKYIFRLHE (SEQ ID NO: 14); KYGKAFDITYVRLKF (SEQ ID NO:
• a method for culturing an isolated population of undifferentiated human embryonic stem cells in chemically defined medium on a synthetic culture surface.
• FIGURE 1 is a flow chart showing an embodiment of a method of making cell culture surfaces.
• FIGURE 2 is an illustration showing a method for making an embodiment of a cell culture surface.
• FIGURE 3 is another illustration showing a method for making an embodiment of a
• FIGURE 4 is a bar graph showing contact angles measured from the base materials.
• FIGURE 5 shows the fluorescence intensity of fluorescently labeled functionalized peptide (having PEO spacer) - grafted to base materials.
• FIGURE 6A-F illustrates fluorescence measurements taken from Rhodamine-labeled functionalized peptide (MAA-PEG 4 -SEQ ID NO:27- NH 2 ) on a GLY-METH base material (FIGURES 6 A-C) and on a HEMA surface (FIGURES 6 D-F).
• FIGURE 7 A-F show photomicrographs of H7 crystal violet-stained human embryonic stem cells cultured on control surfaces MatrigelTM and SynthemaxTM, and on HEMA and Glycerol Methacrylate functionalized peptide-grafted surfaces, VN- MAA grafted to HEMA in FIG 1C, VN-MAA grafted to glycerol in FIG. 7D, VN- PEG4-MAA grafted to HEMA in FIG 7E and VN-PEG4-MAA grafted to Glycerol in FIG. 7F in embodiments of the present invention.
• FIGURE 8A-C are photomicrographs of H7 human embryonic stem cells cultured on control surfaces MG (MatrigelTM, FIG. 8A and SynthemaxTM, FIG. 8B) and on the glycerol VN-PEO4-MAA surface (FIG. 8C) surface in embodiments of the present invention.
• FIGURE 9 shows XPS data showing binding energy of detected oxygen in HEMA surfaces.
• FIGURE 10 shows XPS data showing binding energy of detected oxygen in GLY- METH surfaces.
• the disclosure provides a functionalized peptide having a polymerization moiety (R m ), a spacer moiety (S p ) and a cell adhesive peptide moiety (Cap) and its use in forming a cell culture article suitable for supporting cells in culture.
• the cell culture article formed from the functionalized peptide is suitable for supporting cells in culture in the absence of serum.
• the functionalized peptide has formula R m - S p - C ap wherein R is a polymerization moiety selected from the group consisting of acrylate, methacrylate, acrylamide, methyacrylamide, maleimide fumarate and epoxide and combinations, and m is an integer greater than 1; and, wherein S p is a spacer moiety wherein the spacer moiety comprises polyethylene oxide or polypropylene oxide having the formula 0-CH 2 CHR') ⁇ where R' is H or CH3 and m2 is an integer from 1 to 20, or Xaa n wherein Xaa is any amino acid and n is an integer from 0 to 3, from 0 to 6, from 0 to 10, from 0 to 20 or from 0 to 30, or a combination; and wherein C ap is a peptide comprising a cell adhesive sequence.
• R is a polymerization moiety selected from the group consisting of acrylate, methacrylate, acrylamide, meth
• culturing cells in a scalable fashion requires surfaces that are free of pathogens, relatively inexpensive, stable and reliable, and support long term culture of cells in culture. This is particularly true for cell culture aimed at providing therapeutic cells. That is, cell culture aimed at providing cells which will be introduced or re-introduced into a human for the treatment of disease. While current technology for cell culture includes surfaces that are derived from animal products such as Matrigel , derived from mouse tumor extract, these surfaces are not desirable for support of cells that will be used therapeutically.
• Synthetic cell culture surfaces including surfaces that incorporate synthetic or recombinant proteins or peptides, for example, are suitable for supporting cells that may be introduced into a human for the treatment of disease.
• Synthetic peptides and proteins often include cell adhesive sequences such as RGD.
• Polypeptide sequences are referred to herein by their one letter amino acid codes and by their three letter amino acid codes. These codes may be used interchangeably.
• Synthetic surfaces that reduce the amount of peptide required to support viable cells in culture are desirable, because peptides can expensive, and so surfaces requiring less peptide are less expensive.
• synthetic surfaces that are easy to manufacture, stable in storage, stable through sterilization procedures, and stable through long exposure to aqueous cell culture conditions are also desirable.
• peptides or polypeptides which have been modified or functionalized to carry a polymerization moiety such as an acrylate, methacrylate, acrylamide, methacrylamide, maleimide fumarate or epoxide are provided.
• “functionalized peptide” means peptides which have been modified to incorporate polymerization moieties such as acrylate, methacrylate, acrylamide, methacryalmide, maleimide fumarate or epoxide groups.
• these polymerization moieties can form polymers in the presence of an external energy source such as UV or visible light with an optional catalyst, or by thermal polymerization with an optional catalyst.
• the functionalized peptides or polypeptides may contain a spacer moiety.
• the functionalized peptide is described by formula 1 : [0032] Formula 1 : R m - S p - C ap
• R m is a polymerization moiety, an ⁇ , ⁇ unsaturated group or ethylenically unsaturated group which includes acrylate, methacrylate, acrylamide, methyacrylamide, maleimide, fumarate, or an epoxide, which is capable of polymerizing in the presence of an external energy source, "m" is an integer greater than or equal to 1.
• S p is a spacer.
• S p may be a polyalkylene oxide including for example polyethylene glycol (PEG) or polypropylene glycol (PPG) which are represented by the formula O-CH 2 CHR')m2 where R' is H or CH3 and m2 is an integer from 1 to 20.
• S p may be PEG 2 , PEG4, PEG 6 , PEG 8 , PEG10, PEGi 2 or PPG 2 , PPG 4 , PPG 6 , PPG 8 , PPGi 0 , PPGi 2 Or PPG 20 .
• the surfaces disclosed herein have, in embodiments, glycerol methacrylate and/or
• HEMA hydrophilic base matrices to which functionalized peptides are bound HEMA and glycerol methacrylate have different cell culture characteristics compared to PEG.
• PEG is a non-binding surface. That is, proteins do not adsorb to PEG, and cells to not bind to PEG surfaces.
• PEG in general, has a contact angle of less than 20 degrees.
• HEMA and glycerol methacrylate are not as non-binding as PEG, and the contact angles of surfaces prepared according to embodiments disclosed herein have contact angles of between about 20 degrees and about 60 degrees.
• Sp is PPG or PEG having a functional group.
• the PEG or PPG spacer may have a maleimide, thiol, amine, silane, aldehyde, epoxide, isocyanate, acrylate or carboxyl group.
• the PEG spacer is a Jeffamine, a PEG having an amine functional group.
• the PEG or PPG may be branched.
• the branched PEG or PPO may be a Y- branched or star-PEG or PPG. In embodiments these branched PEG or PPO spacers may allow multiple peptides to be conjugated to a base material through a single functional peptide.
• S p may be an amino acid Xaa n where Xaa is independently any amino acid and n is an integer from 0 to 30, from 0 to 20, from 0 to 10, from 0 to 6 or from 0 to 3.
• the functionalized peptide may be MAA-Lys-Lys-Lys- Lys-Lys-Lys-Lys-VN-Peptide (n-terminal attachment to lysine alpha terminal) or Ac- (Lys-Lys-Lys-Lys-Lys-Lys-MAA)-VN-Peptide (Methacrylate linked) to a series of lysine spacer length sprung from a epsilon lysine side chain.
• the MAA can be attached on the n-terminal of the spacer length or it can be formed on a lysine side chain.
• spacer S p may be a three amino acid sequence such as LysGlyGly or LysTyrGly.
• Xaa n is a series of the same amino acid.
• the spacer S p may be combinations of Xaa n and polyethylene or polypropylene oxide.
• Xaa n may comprise a hydrophilic amino acid such as lysine, glycine, glutamic acid, aspartic acid or arginine amino acid.
• Xaa n may have a terminal lysine or arginine.
• the spacer S p may comprise polyethylene oxide spacer and amino acid spacer in any combination.
• S p may be a hydrophobic spacer such as palmitic acid, stearic acid, lauric acid or hexaethylene diamine (functionalized to allow the hydrophobic moiety to link both to the polymerization moiety and the peptide).
• S p may be carboxyethyl methacrylate.
• the polymerization moiety may attach to the spacer, S p through the polyethylene oxide, through the side chain of an amino acid such as lysine or at the N-terminus of the amino acid.
• Amino acid Xaa n may be acetylated and/or amidated to protect it from degradation. However, if Xaa n is acetylated, the polymerization moiety cannot be bound to Xaa n through the N-terminus of the amino acid.
• C ap is a peptide or polypeptide which has a cell binding or cell adhesive sequence.
• the spacer S p is Xaa n and Xaa n has a terminal lysine.
• Xaa n may be bound to a polymerization moiety R m .
• Xaa n may be (MAA)LysGlyGly or (MAA)LysTyrGly, where MAA is the polymerization moiety methacrylic acid (MAA) bound to Xaa n through the side chain of the terminal lysine amino acid.
• the polymerization moiety may be bound to the N-terminus of the Xaa n amino acid or amino acid chain, if the N- terminus is not acetylated.
• Each functionalized peptide has at least one polymerization moiety, and may have more than one.
• C ap is a peptide or polypeptide having a cell adhesive or cell binding sequence:
• the cell adhesive peptide or cell adhesive polypeptide (which terms are interchangeable) (C ap ) has a cell binding or cell adhesive sequence which may, for example, be an integrin binding sequence or an RGD sequence.
• peptide or polypeptide is an amino acid sequence that may be chemically synthesized or made by recombinant methods.
• peptide or polypeptide is a fragment of a protein, and not a complete protein.
• peptide or polypeptide is not isolated from an animal source.
• peptide or polypeptide may include an amino acid sequence ofYaaiProGlnValThrArgGlyAspValPheThrMetPro (SEQ ID NO: 32), a vitronectin peptide sequence where 1 is an integer from 0 to 3 and where Yaa may be any amino acid or may include, for example, lysine of which the terminal amino acid must be lysine or arginine to accommodate attachment of a polymerizable group.
• the peptide or polypeptide may be cyclic.
• RGDYK(SEQ ID NO:33) may be cyclic c(RGDyK).
• the functionalized peptide has a polymerization moiety which may be a photopolymerizable moiety or a thermal polymerizable moiety.
• This polymerizable moiety may be, for example, an acrylate, methacrylate, acrylamide, methyacrylamide, maleimide fumarate or epoxide moiety.
• the polymerizable moiety may be bound to the Xaa n amino acid sequence through a side chain of the amino acid.
• functionalized peptides have a spacer moiety S p between the cell adhesive peptide (C ap ) and the polymerization moiety R m .
• the spacer may be a hydrophilic spacer, for example, polyethelene oxide (PEO), polyethylene glycol (PEG) or polypropylene oxide (PPO).
• PEO and PEG can be used interchangeably.
• the spacer is PEO 4 .
• the spacer may act to extend the peptide away from the cell culture surface, making the peptide more accessible to cells in culture, and improving the efficiency of the surface for cell culture.
• these hydrophilic spacers may act to repel proteins preventing non-specific absorption to the functionalized peptide.
• a cell adhesive peptide with a spacer such as PEO (polyethylene oxide) in preparing cell culture articles allows for the preparation of such articles using a lower overall concentration of adhesive peptide.
• PEO polyethylene oxide
• These functionalized peptides may be attached, covalently or non-covalently, to the base material.
• Functionalized peptide or polypeptide may be conjugated to the base material at any density, preferably at a density suitable to support culture of cells.
• Functionalized peptide may be conjugated to base material at a density of between about 1 pmol per mm 2 and about 50 pmol per mm 2 of surface, which can be estimated by the surface area of base matrix that is coated in embodiments.
• the functionalized peptide may be present at a density of greater .25 pmol/mm 2 , greater than than .5pmol/mm 2 , greater than 1 pmol/mm 2 , greater than 5 pmol/mm 2 , greater than 6 pmol/mm 2 , greater than 7 pmol/mm 2 , greater than 8 pmol/mm 2 , greater than 9 pmol/mm 2 , greater than 10 pmol/mm 2 , greater than 12 pmol/mm 2 , greater than 15 pmol/mm 2 , or greater than 20 pmol/mm 2 of the base material surface.
• the amount of peptide present can vary depending on the composition of the base material, the thickness of the base material layer and the nature of the polypeptide itself. As discussed below in the Examples, higher densities of peptide may be better able to support attachment and proliferation of undifferentiated stem cells in a chemically defined medium, although other cell types may proliferate more successfully at different peptide densities.
• a base material is provided.
• base material means a polymeric material to which a functionalized peptide is attached.
• the base material is a polymerized or semi-polymerized layer of monomers which provides moieties to allow for the attachment of functionalized peptides.
• the base material may be a hydrophilic base material.
• the hydrophilic base polymeric material may have a low contact angle.
• the hydrophilic base polymeric layer may have a water contact angle of less than 60°, less than 55°, less than 50°, less than 40°, less than 30°, less than 20° or less than 10°. In embodiments, the hydrophilic base polymeric layer has a water contact angle less than 50°.
• the base material is, for example, N-Tris(hydroxymethyl) acrylamide
• cross-linkers can be, for example, N,N'-(l,2-dihydroxyethylene) bisacrylamide, triglycerol diacrylate (glycerol 1,3-diglycerolate diacrylate TGDA), or tetraethylene glycol dimetharcrylate (TEGDMA). Crosslinkers can be interchanged in the different embodiments of the base matrix.
• Acrylate and methacrylate monomers may be synthesized as known in the art or obtained from a commercial vendor, such as Polysciences, Warrington, PA Inc., Sigma Aldrich, Inc., St.Louis, MO and Sartomer, Inc., Exton , PA Polypeptides may be synthesized as known in the art (or alternatively produced through molecular biological techniques) or obtained from a commercial vendor, such as American Peptide, Sunnyvale, CAGenScript Corporation, Piscataway, NJ and Genway Biotech, Inc, San Diego, CA. Spacers may be synthesized as known in the art or obtained from a commercial vendor, such as discrete polyethylene glycol (dPEG) spacers available from Quanta BioDesign, Ltd. Embodiments of the cell culture surface of the present invention are shown in Table 2. 47] Table 2
• the base material may be dispensed onto a substrate, along with a cross-linker.
• the base material may dispensed onto a substrate in lower alcohols such as ethanol.
• the base material may be polymerized, along with a cross-linker, by any polymerizing method.
• the base polymeric material may be polymerized by exposure to UV, visible or thermal energy sources.
• the base polymeric material may be polymerized using a 10s or 30s UV cure time.
• the cure is incomplete. That is, the time of exposure to a polymerizing energy source is insufficient to effect full polymerization of the base polymeric material, resulting in incomplete polymerization or a lower extent of reaction.
• a methacrylate containing base material in the presence of a cross-linker, may be incompletely polymerized, resulting in the presence of methacrylate moieties of these surfaces being available after incomplete polymerization for linking acrylate or methacrylate functionalized RGD containing adhesive peptides.
• the substrate may be any material suitable for culturing cells, including a ceramic substance, a glass, a plastic, a polymer or co-polymer, any combinations thereof, or a coating of one material on another.
• the base material may be flat or shaped.
• Such base materials include glass materials such as soda-lime glass, borosilicate glass, Vycor ® glass, quartz glass; silicon; plastics or polymers, including dendritic polymers, such as poly(vinyl chloride), poly(vinyl alcohol), poly(methyl methacrylate), poly(vinyl acetate-co-maleic anhydride), poly(dimethylsiloxane) monomethacrylate, cyclic olefin polymers, fluorocarbon polymers, polystyrenes, polypropylene, polyethyleneimine; copolymers such as poly(vinyl acetate-co-maleic anhydride), poly(styrene-co-maleic anhydride), poly(ethylene-co-acrylic acid) or derivatives of these or the like.
• dendritic polymers such as poly(vinyl chloride), poly(vinyl alcohol), poly(methyl methacrylate), poly(vinyl acetate-co-maleic anhydride), poly(dimethyl
• cyclic olefin copolymer means a polymer formed from more than one monomer species, where at least one of the monomer species is a cyclic olefin monomer and at least one other monomer species is not a cyclic olefin monomer species.
• cyclic olefin copolymers are formed from ethylene and norbonene monomers.
• Cyclic olefin copolymer resins are commercially available with trade name of TOPAS ® Florence,KY, from Boedeker Plastics, and Inc Zeonor Corporation, Japan.
• the substrate may be treated to enhance retention of the polymer matrix.
• the substrate may be treated with chemical or plasma treatments which provide negative charge, positive charge, create a more hydrophilic surface, or create functional chemical groups that enhance the adhesion of the polymer matrix to the substrate.
• chemical or plasma treatments may include hydrophobic or hydrophilic interactions, steric interactions, affinities or Vander Waal forces.
• FIGURE 1 is a flow chart showing an embodiment of a method of making cell culture surfaces.
• methods for providing cell binding peptides on the surface of a hydrophilic surface by photo-active chemical grafting are provided. These methods include steps of (1) providing a hydrophilic base material to a substrate surface; (2) curing or polymerizing the hydrophilic base material; (3) providing a functionalized peptide to the surface of cured hydrophilic base material; (4) curing or polymerizing the functionalized peptide to the hydrophilic base material; and optionally (5) washing to remove un-reacted monomers.
• the hydrophilic base material and the functionalized peptide may be provided to the surface of a substrate by any means know in the art including liquid dispensing, spin coating, spray coating, or other methods.
• the curing or polymerizing step may be accomplished by any means known in the art, and depending upon the nature of the polymerizing moiety, and may include the introduction of UV, visible or thermal energy to the surface.
• washing may be accomplished by any means known in the art including liquid dispensing and incubating, with or without agitation, where the liquid may be water, a lower alcohol, a lower alcohol diluted in water, or other solvent.
• a composition forming the layer may include one or more additional compounds such as surfactants, wetting agents, photoinitiators, thermal initiators, catalysts and activators. Any suitable polymerization initiator may be employed. One of skill in the art will readily be able to select a suitable initiator, e.g. a radical initiator or a cationic initiator, suitable for use with the monomers. In various embodiments, UV light is used to generate free radical monomers to initiate chain polymerization.
• Any suitable initiator may be used.
• polymerization initiators include organic peroxides, azo compounds, quinones, nitroso compounds, acyl halides, hydrazones, mercapto compounds, pyrylium compounds, imidazoles, chlorotriazines, benzoin, benzoin alkyl ethers, diketones, phenones, or mixtures thereof.
• Suitable commercially available, ultraviolet-activated and visible light-activated photoinitiators have tradenames such as IRGACURE 651, IRGACURE 184, IRGACURE 369, IRGACURE 819, DAROCUR 4265 and DAROCUR 1173 commercially available from Ciba Specialty Chemicals, Tarrytown, N.Y. and LUCIRIN TPO and LUCIRIN TPO-L commercially available from BASF (Charlotte, N.C.)
• Additional initiators may include water soluble azo -initiators that can be used in thermal polymerization including, for example, (VA-044) 2,2'-Azobis[2-(2- imidazolin-2-yl)propane]dihydro chloride; (VA046B) 2,2'-Azobis[2-(2-imidazolin-2- yl)propane]disulfate dehydrate; (VA-50) 2,2'-Azobis(2- methylpropionamidine)dihydrochloride; (VA-057) 2,2'-Azobis[N-(2-carboxyethyl)-2- methylpropionamidine] hydrate; (VA-060) 2,2'-Azobis ⁇ 2-[l-(2-hydroxyethyl)-2- imidazolin-2-yl]propane ⁇ dihydrochloride; (VA-061) 2,2'-Azobis[2-(2-imidazolin-2- yl
• Oil soluble azo -initiators such as (V-70) 2,2'- Azobis(4-methoxy-2.4-dimethyl valeronitrile); (V-65) 2,2'-Azobis(2.4-dimethyl valeronitrile); (V-601) Dimethyl 2,2'-azobis(2-methylpropionate); (V-59) 2,2'- Azobis(2-methylbutyronitrile; (V-40) l,l'-Azobis(cyclohexane-l-carbonitrile) ; (VF- 096) 2,2'-Azobis[N-(2-propenyl)-2-methylpropionamide]; (V-30) l-[(l-cyano-l- methylethyl)azo]formamide; (VAm-110) 2,2'-Azobis(N-butyl-2-methylpropionamide) or (VAm- 111) 2,2'-Azobis(N-cyclohexyl-2-methylpropionamide)
• a photo sensitizer may also be included in a suitable initiator system.
• Representative photo sensitizers have carbonyl groups or tertiary amino groups or mixtures thereof.
• Photo sensitizers having a carbonyl groups include benzophenone, acetophenone, benzil, benzaldehyde, o-chlorobenzaldehyde, xanthone, thioxanthone, 9,10- anthraquinone, and other aromatic ketones.
• Photosensitizers having tertiary amines include methyldiethanolamine, ethyldiethanolamine, triethanolamine, phenylmethyl- ethanolamine, and dimethylaminoethylbenzoate.
• Commercially available photosensitizers include QUANTICURE ITX, QUANTICURE QTX, QUANTICURE PTX, QUANTICURE EPD from Biddle Sawyer Corp, Crawley, England.
• the amount of photo sensitizer or photoinitiator system may vary from about 0.01 to 10% by weight.
• cationic initiators include salts of onium cations, such as arylsulfonium salts, as well as organometallic salts such as ion arene systems.
• the solvent is removed prior to polymerizing.
• the solvent may be removed by any suitable mechanism or process. As described in copending U.S. application serial no. 12/362,782, it has been found that removal of substantially all of the solvent prior to curing, allows for better control of curing kinetics and the amount of monomer converted. When conversion rates of the monomers are increased, waste generation and cytotoxicity are reduced. Using these methods, the resulting base material layer forms a network, but not an interpenetrating network.
• the monomers are polymerized. Whether polymerized in bulk phase (substantially solvent free) or solvent phase, the monomers are polymerized via an appropriate initiation mechanism. Many of such mechanisms are known in the art. For example, temperature may be increased to activate a thermal initiator, photoinitiators may be activated by exposure to appropriate wavelength of light, or the like. According to numerous embodiments, the monomer or monomer mixture is cured using UV light. The curing preferably occurs under inert gas protection, such as nitrogen protection, to prevent oxygen inhibition. Suitable UV light combined with gas protection may increase polymer conversion, insure coating integrity and reduce cytotoxicity.
• inert gas protection such as nitrogen protection
• the hydrophilic base material layer may be washed with solvent one or more times to remove impurities such as unreacted monomers or low molecular weight polymer species.
• the layer is washed with ethanol or an ethanol-water solution, e.g. 70% ethanol, greater than 90% ethanol, greater than 95% ethanol or greater than about 99% ethanol. Washing with a 70% ethanol solvent may not only serve to remove impurities, which may be cytotoxic, but also can serve to sterilize the surface prior to incubation with cells.
• the hydrophilic base material may be provided to a substrate surface and then semi-polymerized. That is, the polymerization process may be controlled or stopped before the base material is fully polymerized. Then a functionalized peptide may be provided to the surface of the semi-polymerized hydrophilic base material on the substrate surface. A second polymerization step may then be applied to polymerize the functionalized peptide to the semi-polymerized hydrophilic base material. The cell culture article may then be washed. The washing step may remove unpolymerized materials.
• FIGURE 2 is an illustration showing a method for making an embodiment of a cell culture surface.
• Tetraethylene glycol dimethacrylate and hydroxyl ethyl methacrylate (HEMA) are applied to a substrate and exposed to UV radiation at 365 nm for 10 seconds or 30 seconds to form a semi-polymerized base material or base layer.
• the functionalized peptide (Ac-Lys-(MAA)-Gly-Gly-Pro-Gln-Val-Thr- Arg-Gly-Asp-Val-Phe-Thr-Met-Pro-NH 2 ) Ac-LyS-(MAA)-SEQ ID NO:28)-NH 2 is then applied to the base layer and exposed to UV radiation at 365 nm for 60 seconds to form the HEMA base material with PEO 4 grafted functionalized peptide.
• R m - S p - C ap R m is methacrylic acid (MAA)
• the cell adhesive peptide (C ap ) is Seq ID NO: 28.
• FIGURE 3 is another illustration showing a method for making an embodiment of a cell culture surface.
• monomers triglycerol diacrylate (Glycerol 1,3- diglycerol diacrylate) and monomethacrylate isomers are deposited on a substrate and exposed to UV radiation at 365 nm for 10 seconds or 30 seconds to form a semi- polymerized base material or base layer.
• the functionalized peptide (MAA)-PEG 4 - Lys-Gly-Gly-Pro-Gln-Val-Thr-Arg-Gly-Asp-Val-Phe-Thr-Met-Pro-NH 2 ) (MAA)- PEG 4 -SEQ ID NO:27)-NH2 is then applied to the base layer and exposed to UV radiation at 365 nm for 60 seconds to form a Glycerol Methacrylate base material with PEO 4 grafted functionalized peptide.
• R m - S p - C ap R m is methacrylic acid (MAA), spacer, S p is present and is PEG 4 , Xaa is absent, and the cell adhesive peptide is Seq ID NO: 27.
• MAA methacrylic acid
• spacer S p is present and is PEG 4
• Xaa is absent
• the cell adhesive peptide is Seq ID NO: 27.
• the cell culture surface may be formed on any surface suitable for cell culture.
• articles suitable for cell culture include single and multi- well plates, such as 6, 12, 96, 384, and 1536 well plates, jars, petri dishes, flasks, beakers, plates, roller bottles, slides, such as chambered and multi-chambered culture slides, tubes, cover slips, bags, membranes, hollow fibers, beads and micro-carriers, cups, spinner bottles, perfusion chambers, bioreactors, CellSTACK ® (Corning, Incorporated) and fermenters.
• Cells may be used for any suitable purpose, including (i) obtaining sufficient amounts of undifferentiated stem cells cultured on a synthetic surface in a chemically defined medium for use in investigational studies or for developing therapeutic uses, (ii) for investigational studies of the cells in culture, (iii) for developing therapeutic uses, (iv) for therapeutic purposes, (v) for studying gene expression, e.g. by creating cDNA libraries, and (vi) for studying drug and toxicity screening.
• Cell culture articles prepared according to embodiments of the methods of the present invention can be effectively presented on the interface of a hydrophilic surface to facilitate growth and proliferation of any relevant cell type, including, for example, stem cells, adult stem cells, Embryonic Stem Cells (ESCs), human Embryonic Stem Cells (hESCs) or Inducible Pluripotent cells (IPCs).
• these cells in culture may be used in therapeutic applications.
• hESC human embryonic stem cells
• the hESC for use in this invention may be obtained from an established cell line.
• human embryonic stem cell lines that have been established include, but are not limited to, Hl, H7, H9, Hl 3 or H14 (available from WiCeIl established by the University of Wisconsin) (Thompson (1998) Science 282:1145 ); hESBGN-01, hESBGN-02, hESBGN-03 (BresaGen, Inc., Athens, GA); HES-I, HES-2, HES-3, HES-4, HES-5, HES-6 (from ES Cell International, Inc., Singapore); HSF-I, HSF-6 (from University of California at San Francisco); I 3, 1 3.2, I 3.3, 1 4, 1 6, I 6.2, J 3, J 3.2 (derived at the Technion-Israel Institute of Technology, Haifa, Israel); UCSF-I and UCSF-2 (Genbacev et al, Fertil.
• Embryonic stem cells used in the invention may also be obtained directly from primary embryonic tissue. Typically this is done using frozen in vitro fertilized eggs at the blastocyst stage, which would otherwise be discarded.
• iPS stem cells include induced primate pluripotent (iPS) stem cells
• OPCs according to the invention may also be differentiated from induced primate pluripotent stem (iPS) cells.
• iPS cells refer to cells, obtained from a juvenile or adult mammal, such as a human, that are genetically modified, e.g., by transfection with one or more appropriate vectors, such that they are reprogrammed to attain the phenotype of a pluripotent stem cell such as an hESC.
• Phenotypic traits attained by these reprogrammed cells include morphology resembling stem cells isolated from a blastocyst as well as surface antigen expression, gene expression and telomerase activity resembling blastocyst derived embryonic stem cells.
• the iPS cells typically have the ability to differentiate into at least one cell type from each of the primary germ layers: ectoderm, endoderm and mesoderm and thus are suitable for differentiation into a variety of cell types.
• the iPS cells like hESC, also form teratomas when injected into immuno -deficient mice, e.g., SCID mice. (Takahashi et al., (2007) Cell 131(5):861; Yu et al., (2007) Science318:5858).
• Embodiments of the present invention provide for efficient techniques for decorating surfaces with cell binding adhesive ligands such as peptides in a cost effective manner and facile manufacturing processes leading to overall significant reduction in manufacturing costs.
• the surfaces are useful surfaces for culturing cells, including human embryonic stem cells in the pluripotent (having more than one potential outcome) state using chemically defined media.
• chemically defined media, in combination with synthetic surfaces in embodiments of the present invention is important because the use of serum introduces undefined factors into cell culture which may be detrimental to cultured cells intended for therapeutic uses.
• FIGURE 4 is a bar graph showing contact angles measured from the base materials (1-4) shown in Table 2. In embodiments, the surfaces have a water contact angle less than 60 degrees, between 15 and 60 degrees, between 20 and 60 degrees, greater than 15 degrees or greater than 20 degrees.
• FIGURE 5 shows the fluorescence intensity of peptide (having PEO spacer).
• FIGURE 5 shows fluorescence of MAA-PEO 4 -LyS-GIy-GIy-PrO-GIn- Val-Thr-Arg-Gly- Asp- Val-Phe- Thr-Met-Pro-NH 2 (MAA-PEO 4 -SEQ ID NO:27-NH 2 ) doped with 0.2% of rhodamine labeled version conjugated to six different base matrices, all of which are described in Table 2: N-Tris-Acrylamide (1) Glycerol Methacrylate (2), Sorbitol (3), Pentaerythritol (4), Poly-Serine (5) and HEMA (6).
• the more hydrophilic glycerol base matrix and NTRIS -Methacrylate surfaces had greater peptide grafting efficiency than the less hydrophilic HEMA and pentaerythritol methacrylate base matrix.
• the PEO 4 spacer provided greater accessibility and therefore render improved peptide grafting efficiency over the peptide without spacer. It was observed that surfaces with lower contact angle provided for more efficient spreading of the functionalized peptide monomer on the base, and lower consumption of the photo-active peptide.
• contact angle refers to the initial contact angle of water on the substrate, without peptide.
• a functionalized peptide comprising a composition of the formula R m - S p - C ap wherein R is a polymerization moiety selected from the group consisting of acrylate, methacrylate, acrylamide, methyacrylamide, maleimide fumarate and epoxide and combinations, and m is an integer greater than 1 ; and, wherein S p is a spacer moiety wherein the spacer moiety comprises polyethylene oxide or polypropylene oxide having the formula 0-CH 2 CHR') ⁇ where R' is H or CH3 and m2 is an integer from 1 to 20, or Xaa n wherein Xaa is independently any amino acid and n is an integer from 0 to 3, or a combination; and wherein C ap is a peptide comprising a cell adhesive sequence is provided.
• the functionalized peptide of aspect 1 wherein S p is a Lys or Arg amino acid is provided.
• the functionalized peptide of aspect lor 2 wherein the cell adhesive peptide (C ap ) comprises the sequence: KGGGQKCIVQTTSWSQCSKS (SEQ ID NO:1); GGGQKCIVQTTSWSQCSKS(SEQ ID NO:2); KYGLALERKDHSG (SEQ ID NO:3); YGLALERKDHSG (SEQ ID NO:4); KGGSINNNRWHSIYITRFGNMGS (SEQ ID NO:5); GGSINNNRWHSIYLTRFGNMGS (SEQ ID NO:6); KGGTWYKIAFQRNRK (SEQ ID NO: 7); GGTWYKI AFQRNRK (SEQ ID NO: 8); KGGTSIKIRGTYSER (SEQ ID NO:9); GGTSIKIRGTYSER (SEQ ID NO: 10); KYGTD
• the functionalized peptide of aspect 1 wherein the cell adhesive peptide (Cap) comprises KGGPQVTRGDVFTMP (SEQ ID NO:27) or GGPQVTRGDVFTMP(SEQ ID NO:28) is provided.
• the functionalized peptide of aspects 1-4 are provided wherein the polymerization moiety (R m ) comprises an acrylate or a methacrylate.
• a cell culture article comprising the functionalized peptide if claim 1 covalently linked to a hydrophilic polymeric base material, wherein the hydrophilic polymeric base material comprises N-Tris(hydroxymethyl)acrylamide (ACRYLNTRIS-OH) and N,N'-(l,2-dihydroxyethylene)bisacrylamide copolymer, glyceryl monomethacrylate and glycerol 1,3-diglycerolate diacrylate copolymer (GLY-METH) or poly(serine)methacrylate and glycerol 1,3-diglycerolate diacryate copolymer (SER-METH), hydro xyethylmethylacrylate (HEMA), or acrylamide (ACRYL) polymers and copolymers and optionally hydrophilic crosslinking materials such as, N,N'-(l,2-dihydroxyethylene)bisacrylamide, glycerol 1,3-diglycerolate diacrylate, or combinations thereof is provided.
• the cell culture article of aspect 7 is provided wherein the cell culture article has a contact angle of less than 50°. In an aspect, the cell culture article of aspect 7 is provided wherein the hydrophilic polymeric base material has a contact angle greater than 20°. In an aspect, the cell culture article of aspect 7 is provided wherein the hydrophilic polymeric base material has a contact angle between 20° and 60°. In an aspect, the cell culture article of aspect 7 is provided wherein the hydrophilic polymeric base material has a contact angle between 20° and 60°. In an aspect (9), the cell culture article of aspect 7 or 8 is provided wherein the cell adhesive peptide is selected from the group consisting of KGGGQKCIVQTTSWSQCSKS (SEQ ID NO:1);
• GGGQKCIVQTTSWSQCSKS (SEQ ID NO:2); KYGLALERKDHSG (SEQ ID NO:3); YGLALERKDHSG (SEQ ID NO:4); KGGSINNNRWHSIYITRFGNMGS (SEQ ID NO:5); GGSINNNRWHSIYITRFGNMGS (SEQ ID NO:6);
• GRGDSPK SEQ ID NO:29
• KGGAVTGRGDSPASS SEQ ID NO:30
• GGAVTGRGDSPASS SEQ ID NO:31
• YaaiPQVTRGNVFTMP SEQ ID ;
• the cell culture article of any one of aspects 7 - 9 is provided wherein the spacer S m comprises PEO 4 .
• a method of making the cell culture article of claim 7 comprising the steps of: providing a hydrophilic base material to a substrate surface; semi-polymerizing the hydrophilic base material; providing a functionalized peptide to the surface of the semi-polymerized hydrophilic base material; polymerizing the functionalized peptide to the semi-polymerized hydrophilic base material; and, optionally washing is provided.
• the hydrophilic base material comprises N-Tris(hydroxymethyl)acrylamide
• the method of aspect 11 or 12 is provided wherein the semi-polymerized hydrophilic base material has a water contact angle of less than 50°.
• the method of any one of aspects 11 - 14 is provided wherein R m comprises an acrylate or methacrylate.
• R m comprises an acrylate or methacrylate.
• S p comprises PEO4.
• C ap is a cell adhesive peptide selected from the group consisting of: KGGGQKCIVQTTSWSQCSKS (SEQ ID NO:1);
• GGGQKCIVQTTSWSQCSKS (SEQ ID NO:2); KYGLALERKDHSG (SEQ ID NO:3); YGLALERKDHSG (SEQ ID NO:4); KGGSINNNRWHSIYITRFGNMGS (SEQ ID NO:5); GGSINNNRWHSIYLTRFGNMGS (SEQ ID NO:6);
• KGGTWYKIAFQRNRK (SEQ ID NO:7); GGTWYKIAFQRNRK (SEQ ID NO:8); KGGTSIKIRGTYSER (SEQ ID NO:9); GGTSIKIRGTYSER (SEQ ID NO: 10); KYGTDIRVTLNRLNTF (SEQ ID NO: 11); YGTDIRVTLNRLNTF (SEQ ID NO:
• GRGDSPK SEQ ID NO:29
• KGGAVTGRGDSPASS SEQ ID NO:30
• GGAVTGRGDSPASS SEQ ID NO:31
• YaaiPQVTRGNVFTMP SEQ ID NO:32
• RGDYK SEQ ID NO:33
• GMMA Glycerol monomethacrylate, TEGDMA - Tetraethyleneglycol dimethacrylate, HEMA - 2-hydroxyethyl methacrylate
• ACRYLNTRIS N- Tris(hydroxymethyl)acrylamide + N, N'-(l,2-dihydroxyethylene)bisacrylamide, also NTRIS-ACRYLAMIDE
• SER-METH (Poly(serine)4Methacrylate + Glycerol 1,3- Diglycerolate
• GLY-METH (Glycerol monomethacrylate + Glycerol 1,3- Diglycerolate Dimethacrylate, also shown as Glycerol in Table 2), (2-hydroxyethyl methacrylate + Tetraethyleneglycol dimethacrylate), EtOH - Ethanol, 1-819, Darocur 1173.
• Photoinitiators Irgacure-819 Phosphine oxide, phenyl bis(2,4,6-trimethyl benzoyl) and Darocur 1173 (2-hydroxy-2-methyl- 1 -phenyl- 1-propanone) used in the free radical polymerization of the formulations were obtained from Ciba Specialty Chemicals (Newport Delaware) and used without any further purification.
• Hydrophilic crosslinkers, tetraethylene glycol dimethacrylate (86680), (454982) and glycerol 1,3-diglycerol diacrylate (475807) N, N'-(l,2- dihydroxyethylene)bisacrylamide (37474) were all purchased from Sigma-Aldrich in the purity as described in product specification sheet.
• Hydrophilic monomers 2- hydroxyethylmethacrylate, +99% (477028) and N-Tris(hydroxymethyl)acrylamide were purchased from Sigma-Aldrich while the other hydrophilic monomer used in the formulations, glycerol monomethacrylate isomers (04180) was purchased from Polysciences Incorporated without further purification.
• Poly(Serine) 4 Methacrylate used as a hydrophilic methacrylate functionalized polyamino acid along with adhesive peptides Ac-Lys(MAA)-Gly-Gly-Pro-Gln-Val-Thr-Arg-Gly-Asp-Val-Phe-Thr-Met- PrO-NH 2 (SEQ ID NO:27) and (MAA-PEO 4 -VN): MAA-PEO 4 -Lys-Gly-Gly-Pro- Gln-Val-Thr-Arg-Gly-Asp-Val-Phe-Thr-Met-Pro-NH 2 (SEQ ID NO:27) were synthesized by American Peptide, Sunnyvale, CA by the following processes.
• Reagents for coupling and cleavage were purchased from Aldrich. Solvents were purchased from Fisher Scientific.
• the peptide chain was assembled on resin by repetitive removal of the Fmoc protecting group and coupling of protected amino acid. DIC and HOBt were used as coupling reagents and NMM was used as base. 20% piperidine in DMF was used as de-Fmoc-reagent.
• Methacrylic acid (MAA) was coupled on the side chain of Lysine after Ivdde was removed by 2% Hydrazine in DMF. After the last coupling, resin was treated with TFA/TIS/H2O (95:3:2, v/v/v) for cleavage and removal of the side chain protecting groups.
• MAA-PEO 4 -VN MAA-PEO 4 -LyS-GIy-GIy-PrO-GIn- Val-Thr-Arg- Gly-Asp-Val-Phe-Thr-Met-Pro-NH 2 (SEQ ID NO:27): The peptide was synthesized by American Peptide Sunnyvale, CA on lmmol Fmoc-Rink Amide resin via Fmoc chemistry. Protecting groups used for amino acids are: t-Butyl group for and Asp and Thr, Trt group for GIn, Pbf for Arg, Boc for Lys.
• Fmoc protected amino acids were purchased from EMD Biosciences; Fmoc-PEG4-OH was purchased from Quanta Biodesign. Reagents for coupling and cleavage were purchased from Aldrich. Solvents were purchased from Fisher Scientific. The peptide chain was assembled on resin by repetitive removal of the Fmoc protecting group and coupling of protected amino acid. HBTU and HOBt were used as coupling reagents and NMM was used as base. 20% piperidine in DMF was used as de-Fmoc-reagent. Methacrylic acid (MAA) was coupled to the amino group of PEG4 after removal of the Fmoc protecting group.
• MAA Methacrylic acid
• the other grafting peptide VN-MAA Lys(MAA)-Gly-Gly-Pro-Gln-Val-Thr-Arg-Gly-Asp-Val-Phe-Thr-Met-Pro-NH 2 (Lys- Maa-SEQ ID NO:27-NH 2 ) in 8.74 mg (0.5 mM) was also prepared in similar fashion.
• a semi-automated pipettor was used to dispense, onto tacky surface 200 ⁇ L or 500 ⁇ L of stock solution of MAA-PEG 4 -Lys-Gly-Gly-Pro- Gln-Val-Thr-Arg-Gly-Asp-Val-Phe-Thr-Met-Pro-NH 2 (MAA-PEG 4 -SEQ ID NO:27- NH 2 ), and Lys(MAA)-Gly-Gly-Pro-Gln-Val-Thr-Arg-Gly-Asp-Val-Phe-Thr-Met- PrO-NH 2 (Lys-MAA-SEQ ID NO:27-NH 2 ) into respective 6-wp per plate.
• the solution was allowed to spread for 15 minutes, then cured for 1 minute, then washed for 1 h in ethanol followed by 1 h in PBS buffer, then rinsed for 1 minute with DI water.
• the base hydrophilic matrix only was applied to a 6wp substrate and cured for 10 or 30 seconds. Contact angle measurements were made on the base substrate.
• 10 ⁇ L of Rhodamine-labeled peptide was added to 1 ml of peptide-methacrylate and coated on their respective hydrophilic base matrix. Surfaces were then cured for 60 seconds and surfaces were scanned before and after washing.
• Measurement of Contact Angle The water contact angle measurements were obtained on Rame-Hart goniometer (Rame-Hart Instrument Company, 95 Allen Street, Netcong, NJ, 07857-0400) using dl water and measured within one minute after the water was placed on the surface.
• FIGURE 6 illustrates fluorescence measurements taken from Rhodamine-labeled functionalized peptide (MAA-PEG 4 -SEQ ID NO:27- NH 2 ) on a GLY-METH base material (FIGURES 6 A-C) and on a HEMA surface (FIGURES 6 D-F).
• the peptide grafting efficiency was 3X higher for the GLY-METH base material, which is more hydrophilic, compared to the HEMA base material.
• FIGURE 7 shows photomicrographs of H7 human embryonic stem cells cultured on control surfaces Matrigel available from BD, Franklin Lakes NJ, (FIGURE 7A), SynthemaxTM available from Corning Incorporated, Corning, NY (FIGURE 7B), VN- MAA functionalized peptide on a HEMA base (FIGURE 7C), VN-MAA functionalized peptide on a glycerol methacrylate (GLY METH) base (FIGURE 7D), VN-PEO4-MAA on a HEMA base (FIGURE 7E) and VN-PEO 4 -MAA on a glycerol methacrylate (GLY METH) base (FIGURE 7F).
• FIGURE 7A shows photomicrographs of H7 human embryonic stem cells cultured on control surfaces Matrigel available from BD, Franklin Lakes NJ, (FIGURE 7A), SynthemaxTM available from Corning Incorporated, Corning, NY (FIGURE 7B), VN- MAA
• Functionalized peptide grafted to GLY-METH base material showed human stem cell growth and morphology similar to control after four days in culture, with both the (Lys-MAA-SEQ ID NO:27-NH 2 ) and the (MAA-PEG 4 -SEQ ID NO:27- NH 2 ) functionalized peptides, compared to the less hydrophilic HEMA surface.
• GLYC-METH (37.4°) resulted in higher density of MAA-PEG 4 -LyS-GIy-GIy-PrO- Gln-Val-Thr-Arg-Gly-Asp-Val-Phe-Thr-Met-Pro-NH 2 (MAA-PEG 4 -SEQ ID NO:27- NH 2 ) grafting than its HEMA counterpart with contact angle of (54.9°) and is considered a preferred embodiment for cell culturing of H7 hESCs.
• different cells may survive in cell culture on base materials having higher or lower contact angles, depending upon the cell culture preferences of that particular cell type.
• a functionalized peptide, or peptide methacrylate with PEO 4 spacer length resulted in higher grafting efficiency than one without a PEO 4 spacer.
• the surfaces with more efficient peptide grafting and therefore higher peptide density were able to show significant improvement in cell adhesion and spreading of H7 Human Embryonic Stem Cells (hESCs) over 4 days of culturing.
• hESCs H7 Human Embryonic Stem Cells
• Mass transport in and on the interface of the matrix may allow for efficient spreading during grafting of methacrylate functionalized peptides while the inclusion of a PEO 4 spacer on peptide may allow for providing ligand accessibility and proper orientation for putative cell attachment and spreading of human embryonic stem cells (hESCs).
• hESCs human embryonic stem cells
• Increasing the hydrophilic nature of the base material may be responsible for several occurrences: 1) mass transport of methacrylate functionalized peptides are improved over the surface, 2) un-reacted methacrylates from deliberate under-curing of the base matrix are rendered more mobile on the surface interface and therefore have greater accessibility for connecting with methacrylates; and, 3) less volume of solution may be required to facilitate grafting due to hydrophilic tunability of these surfaces. Additional studies (not presented) conducted using different chemistries to impart hydrophilicity show a continued trend of increase grafting efficiency with decreasing contact angle.
• FIGURE 8 shows photomicrographs of H7 human embryonic stem cells grown on control surfaces MG (MatrigelTM), (FIGURE 8A), SynthemaxTM (FIGURE 8B), and the glycerol-methacrylate VN-PE04-MAA (GLY-METH) for four days.
• FIGURE 8 illustrates that the morphology of H7 hESC cells cultured on embodiments of the cell culture surface of the present invention is comparable to MatrigelTM and SynthemaxTM.
• FIGURE 9 shows XPS data showing binding energy of detected oxygen in HEMA surfaces.
• the atomic composition of the top 2-6 nm of the surfaces is shown in Tables 3 and 4. These data were collected based on only 2 different scanned areas, and using a lower resolution pass energy of ⁇ 45 eV because there was a need to collect the data quickly in order to minimize the chance of beam or vacuum damage.
• the results of the XPS analysis for the HEMA surface are shown in Table 3.
• FIGURE 10 shows XPS data showing binding energy of detected oxygen in GLY- METH surfaces. The results of XPS analysis are shown in Table 4:
• XPS showed that the surface with a higher contact angle (acrylated glycol) displayed higher oxygen content.
• the GLYC-METH surface showed a higher C-O peak than the HEMA surface. This is consistent with the presence of a greater number of OH groups on the surface of the GLY-METH surface.
• XPS also showed that these two surfaces contain different ratios of oxygen-containing functionalities.
• the high oxygen content of the glycerol surfaces is shown by the increased oxygen containing groups on the surface that is responsible for driving the higher surface energy (low/receding) contact angle that facilitates spreading of methacrylate functionalized peptide for grafting.

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